PURPOSES:To investigate the time of neuronie apoptosis in the retinas of Imman fetuses,and its relations with neuronie proliferation and differentiation, METHODS:The retinas of 27 human fetuses from 8th to 38th week of R,~til-
ization age and 3 adults were studied by TdT-mediated dUTP nick end labelling(TUNEL) method.
RESULTS:Tbe nuctei of labeled apoptotic cells were charaeterised by nuclear marginization,ehromatln condensation and cleseent shape,and some
apoptotie bodies were visible in the specimens. The apoptosis of neuroepithelium of fetal rclina took place during 8th to 18th week, Apoptosis of ganglion cells were observed from 1256 to 18th week. The apoptos[s of pholorec, plors were formd from 14th to 2Ist week ,while thai of bipolar neurones and M~ller cells were found from ldth to 28th week. No apoptosb of ocstones were observed in the retinas after 28th week of fertilization age and within the retinas of adults.
CONCLUSION:The proliferating cells of neuroepithelium and Ihe neurones which just differetiated from fetal retina might partly undergo apoptosis. The time of apoptosls of differentiated neurones was consistent with the time of the synapses formation between neurones and their targel cells.
（Chin J Ocul Fundus Dis,1997,13:67 -69 ）
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
Objective To observe the effects of minocycline to the viability and apoptosis of ratprime;s retinal neuron cells (RNC) under pressure, and to investigate the neuroprotective mechanisms of minocycline against the RNC damage. Methods Establish a model of ratprime;s RNs under pressure cultured in vitro, the protective effect of minocycline is observed by different methods, including observing the morphology of the cells, evaluating the cellsprime; viability by methyl thiazolyl tetrazolium (MTT) colorimetry assay, and detecting the cellular apoptosis with acridine orange/ethidium bromide (AO/EB) double staining by fluorescence microscopy. Immunocytochemistry was used to detect the expression of iNOS and caspase-3 in the cells. Results Obvious morphology changes of RNC were found in cells under pressure compared with the control; the viability of RNC decreased and cellular apoptosis was found in 53.93% cells. The cellular morphology improved in the cells treated by 20 mu;mol/L minocycline, the cellular viability significantly increased, and the cellular apoptosis was found in 17.29% cells. In addition, the expression of iNOS and caspase3 in the treated cells decreased compared with which in the pressured group. Conclusion Minocycline with a certain concentration can effectively inhibit pressureinduced damage and apoptosis of RNC of rats, and the inhibitory effect on expression of iNOS and capases-3 may be the underlying mechanism.
Objective
To observe the ultrastructural characteristics of human retinal progenitor cells cultured in vitro.
Methods
Six 5-month-old human fetuses（12 eyes）without eye diseases were selected.
Retinal
progenitor cells from the retina of one eye of each fetus were cultured in vitro，and observed by transmission electronic microscopy（TEM）; while those from the other eye were directly observed by TEM. Results Abundant heterochromatin were found in the karyon of 5-month embryonic retinal neuroepithelial cells，and the figure of the karyons was irregular.A few scattered initial cells were seen in retinal neuroepithelial layer with large karyon，smooth surface，abundant euchromatin，and distinct nucleolus.The human retinal progenitor cells cultured in vitro had the same ultrastructural characteristics as the initial cells:with huge karyon which almost occupied the whole cell，little cytoplasm，distint nucleolus，abundant euchromatin，and little heterochromatin.The cells clung to each other in the neural globoid cell mass.The size of the outer cells was large，and karyokinesis could be found.
Conclusion
The cultured human retinal progenitor cells are provided with the same ultrastructure characteristics as the initial cells.
(Chin J Ocul Fundus Dis, 2006, 22: 185-187)
Objective To investigate the characteristics and possibility of using an image analyzer-aided method to count axotomized retinal ganglion cells (RGCs). Methods The left optic nerves of 18 rats were transected intraorbitally and a piece of gelform soaked in 5% fluorogold was applied to the ocular stump to retrogradely label the surviving RGCs. All animals were executed 2, 7 or 14 d ays after the operation (n=6 for each time point), respectively. The left retinae were removed, post-fixed and whole-mounted on the slides. The numbers of labeled RGCs were counted using both the conventional sampling method and image analysis, and compared statistically between the two methods.Results The number of surviving RGCs decreased sharply［(12 0663±9 089), (59 285±17 071) and (17 802±19 8 4) cells/mm2 for image analyzer-aided method, and (118 237±7 898), (57 648±14 533) and (18 070±1 461) cells/mm2 for conventional sampling method］when the survival time increased from 2 to 7 and 14 days. No significant difference was detected between the two groups at any corresponding time points.Conclusion The image analyzer-aided method is convenient, objective and reproducible, which can be used in the studies where counting RGCs is needed. (Chin J Ocul Fundus Dis，2003，19：333-404)
Objective
To study the effects of neonatol rabbit Schwann cells(SC) on repair of optic contusion in adult rabbits.
Methods
24 h after the adult rabbit optic nerves was contused,0.1 ml of SC suspension (group A) and saline water (group B) were injected into the vitreous of injured eyes respectively.All the animals were studied by retinal ganglion cell (RGC) and axon counting,flash visual evoked potential (FVEP) tests at various intervals after injury.
Results
At the 4th week after injury,the number of RGC was (19.89plusmn;3.79)/mm in group A and (12.67plusmn;4.12)/mm in group B,and the density of axons was (94.569plusmn;793)/mm2 in group A and (36.085plusmn;285)/mm2 in group B.There was dramatical difference between group A and B (Plt;0.01).The amplitude of FVEP wave of group A increased from 48% to 88% on the 3rd day after injury,and still dept 78% at the 8th week and group A was significantly higher than group B at various intervals (Plt;0.01).
Conclusion
SC are effective in promoting the repair of optic nerve contusion by increasing the survival rate of RGC,rescuing axons from degeneration,and dramatically promoting the function of the optic nerve.
(Chin J Ocul Fundus Dis,2000,16:91-93)
Object ive To summa r i z e the advanc ement of cytoske l e ton and axon outgrowth of neuron. Methods The recent l iterature concerning cytoskeleton and axon outgrowth of neuron was reviewed and summarized. Results The actin filaments and microtubules in neuron were highly polarized and dynamic structures confined to the ti ps of axons and the reci procal interactions between these two major cytoskeletal polymers was also dynamic. Attractive or a repulsive cue whose final common path of action was the growth cone cytoskeleton mediated the growth of axons of neuron by intracellular signaling cascades. Regulating the actin filament and microtubule dynamics as well as their interactions in growth cones played a key role in neurite outgrowth and axon guidance. Rho-GTPases and glycogen synthase kinase 3β (GSK-3β), the two major intracellular signal ing pathways had emerged in recent years as candidates for regulating the dynamics of actin filaments and microtubules. Conclusion The axon outgrowth and guidance depend on well-coordinated cytoskeletal and reciprocal interaction dynamics which also mediate axon regeneration after spinal cord injury. Regulating activity of Rho-GTPases and GSK- 3β simultaneously may acts as key role to regulate the dynamics of cytoskeletal and to determine axon outgrowth.
ObjectiveTo study the inducting differentiation effect of the sciatic nerve extracts on rabbit adipose-derived stem cells (ADSCs) in vitro.
MethodsThe ADSCs were isolated from 2 healthy 4-month-old New Zealand rabbits (weighing, 2.0-2.5 kg) and cultured to passage 3, which were pretreated with 10 ng/mL basic fibroblast growth factor (bFGF) for 24 hours before induction. Then the induction media containing the extracts of normal sciatic nerve (group B) and injured sciatic nerve at 3, 7, and 14 days (group C, group D, and group E) were used, and D-Hank was used in group A as blank control group. The morphological changes of the cells were observed. At 7 days of induction, the gene expressions of neuron-specific enolase (NSE), nestin (NES), and S-100 were detected by real-time fluorescent quantitative PCR. The S-100 protein expression was tested by immunocytochemical staining.
ResultsAt 4 days after induction, some ADSCs of groups C, D, and E showed the morphology of Schwann-like cells or neuron-like cells, the change of group D was more obvious; and the ADSCs of group A and B had no obvious change, which were still spindle. The S-100 immunocytochemical staining showed positive expression in groups C, D, and E (more obvious in group D) and negative expression in groups A and B. The gene expression of S-100 displayed time-dependent increases in groups C and D, which was significantly higher than that of groups A, B, and E (P<0.05), but no significant difference was found between groups C and D (P>0.05). The gene expression of NSE showed the same tendency to S-100, which reached the peak in group D; the gene expression of NSE in groups D and E was significantly higher than that of groups A, B, and C (P<0.05), and groups D and E showed significant difference (P<0.05). However, the gene expression of Nestin showed no significant difference among different groups (P>0.05).
ConclusionThe ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; and the early stage (3-7 days) after injury is the best time for stem cell transplantation.
Objective To observe the serumlevel of neuron-specific enolase( NSE) in patients with pulmonary encephalopathy and its changes after treatment with mechanical ventilation. Methods Twentyone patients with pulmonary encephalopathy were enrolled. Glasgow coma scale( GCS) , serumNSE level, and arterial blood gas were evaluated at three time-points: before mechanical ventilation, after 12 hours mechanical ventilation, and the moment of consciousness. Results 18 patients recovered consciousness, and 3 patients remained in persistent coma and died. GCS and arterial blood gas improved obviously after 12 hours mechanical ventilation. Meanwhile, the serumNSE concentration decreased significantly after 12 hours mechanical ventilation [ ( 24. 54 ±6. 65) μg/L] and at the moment of consciousness [ ( 14. 19 ±2. 91) μg/L] compared with before mechanical ventilation( P lt; 0. 05, P lt; 0. 01) . Conclusion Dynamic measurment of serumNSE may be a useful biomarker for assessing the severity of cerebral injury and predicting prognosis.
Objective
To investigate the glutamate toxicity on inner stratum retinal neurons(ISRN) and the neurotoxicity quantity-efficacy relation.
Method
Retinal explants obtained from 30 neonatal mices were implanted into two pieces of 24-well culture plates (48 wells). The 48 wells were divided into three groups: control group, glutamate exposure 24 h group, and glutamate exposure with further lasting 6 h group. The retinal explants were sectioned, and then stained with HE after 24 h in vitro. The cells in retinal ganglion cells (RGCs) layer and inner nuclear layer (INL) were analyzed by light microscope at 1 000times; magnification , and the number of normal morphological cells was counted under three 1 000times; magnificat ion fields.
Results
Some cells in ISRN (include RGCs and INL c ells) showed pykno tic nuclei and necrosis after 24 h in control culture. Glutamate exposure 24 h group:at the 2 mmol and 4 mmol concentrations of glutamate, the situation of the normal morphological cells in ISRN had no difference from that of the control group (Pgt;0.05). At the concentration of glutamate more than or equal to 6 mmol, the number of normal morphological cells in ISRN was significantly less than that of the control group (Plt;0.05), and with the increase of glutamate concentration, the number of normal morphological cells was reduced. Glutamate exposure with fur ther lasting 6 h group: at the concentration of glutamate equal to 6 mmol, the n umber of normal morphological cells in INL was significantly less than that of the control group (Plt;0.05), while the number of normal morphological cells in RGCs layer had no difference between two groups (Pgt;0.05). At the concentration of glutamate more than or equal to 8 mmol, the number of normal morphological cels in RGC s layer and INL was significantly less than that of the control group (Plt;0.05 ).
Conclusion
Glutamate has the neurotoxicity for ISRN in vitro, and the effect is dose-dependant.
(Chin J Ocul Fundus Dis, 2001,17:311-314)
